Multiple Sclerosis (MS) is a T cell mediated autoimmune central nervous system demyelinating disease resulting from poorly understood genetic and environmental interactions. The deficiency or dysregulation of N-glycan biosynthesis plays a critical role in the pathogenesis of autoimmunity. The Hexosamine and N-glycan pathways conditionally regulate autoimmunity by titrating T cell activation, TH1 differentiation and susceptibility to spontaneous autoimmune demyelinating disease in mice. 1a,25-Dihydroxyvitamin D3 inhibits T cell activation, TH1 differentiation and Experimental Autoimmune Encephalomyelitis (EAE) in mice by an unknown mechanism and is an environmental factor whose deficiency is associated with MS. T cell [unreadable]1,6GlcNAc-branched N-glycans are enhanced by 1a,25-Vitamin D3 in vitro and reduced in vivo by dietary deficiency of Vitamin D3. Blocking [unreadable]1,6GlcNAc-branched N-glycan expression reverses 1a,25-Vitamin D3 induced T cell hypo-proliferation, indicating 1a,25-Vitamin D3 negatively regulates T cell proliferation through [unreadable]1,6GlcNAc-branched N-glycans. These data identify Vitamin D3 as an important regulator of T cell [unreadable]1,6GlcNAc branching and suggest that Vitamin D3 negatively regulates T cell function and autoimmune disease via this mechanism. To examine this hypothesis, three Specific Aims are proposed. Specific Aim #1 will investigate the mechanism by which Vitamin D3 promotes [unreadable]1 ,6GlcNAc-branching. Specific Aim #2 will determine if Vitamin D3 regulates T cell function by increasing [unreadable]1,6GlcNAc-branching. Specific Aim #3 will determine if Vitamin D3 regulates N-glycan processing in vivo and autoimmune disease by increasing [unreadable]1,6 GlcNAc-branching. This work will establish if this therapeutic approach should be considered for the treatment of MS. Furthermore, identifying the mechanisms that regulate immune tolerance and autoimmunity is a key goal in the field of immunology. MS afflicts ~300,000 people in the USA and results in variable neurological dysfunction such as blindness, paralysis and cognitive dysfunction. MS results from poorly understood genetic and environmental factors. This work seeks to investigate mechanisms promoting disease and avenues for possible therapeutics.